Docking and fusion of synaptic vesicles in cell-free model system of exocytosis.
The present study involves the testing and characterization of synaptic vesicle (SV) docking and fusion as the steps of exocytosis using two different approaches in vitro. The interaction of SVs was determined by the changing of particles size in suspensions by the method of dynamic light scattering (DLS). Fluorescence assay is represented for studying the mechanism of SV membrane fusion. The sizes of membrane particles were shown to increase in the medium containing cytoplasmic proteins of synaptosomes. Therefore, the cytosolic proteins are suggested to promote the SVs into close proximity where they may become stably bound or docked. The specific effect of synaptosomal cytosolic proteins on the interaction of SVs in the cell-free system was demonstrated. The incubation of SVs with liver cytosol proteins or in the bovine serum albumin solution did not lead to the enlargement of the particles size. The fusion reaction of the SVs membranes occurred within the micromolar range of Ca(2+) concentrations. Our studies have shown that in vitro process of exocytosis can be divided into Ca(2+)-independent step, termed docking and followed by fusion step that is triggered by Ca(2+). The role of cytosolic proteins of synaptosomes in docking and fusion of SVs in cell-free system was further confirmed.
Trikash
I O
IO
National Academy of Sciences of Ukraine, Palladin Institute of Biochemistry, Department of Neurochemistry, Leontovich st. 9, 01601 Kiev, Ukraine. trikash@biochem.kiev.ua
Volynets
G P
GP
Remenyak
O V
OV
Gorchev
V F
VF
D000818:Animals; D001681:Biological Assay; D002118:Calcium / Q000502:physiology; D020013:Calcium Signaling / Q000502:physiology; D002474:Cell-Free System / Q000502:physiology / Q000648:ultrastructure; D002490:Central Nervous System / Q000378:metabolism / Q000648:ultrastructure; D003600:Cytosol / Q000378:metabolism / Q000648:ultrastructure; D005089:Exocytosis / Q000502:physiology*; D005456:Fluorescent Dyes; D008081:Liposomes / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology*; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000378:metabolism; D017729:Presynaptic Terminals / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence; D013570:Synaptic Membranes / Q000378:metabolism* / Q000648:ultrastructure; D009435:Synaptic Transmission / Q000502:physiology*; D013572:Synaptic Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D013574:Synaptosomes
D005456:Fluorescent Dyes; D008081:Liposomes; D009419:Nerve Tissue Proteins; D002118:Calcium